Aurelija Vaitkuviene

Laser-induced fluorescence studies of premalignant and benign lesions in the female genital tract

Laser-induced fluorescence (LIF) was studied in vivo from premalignant and benign lesions in the female genital tract, in particular the cervix. The aim of the study was to investigate the possibilities to differentiate cervical intraepithelial neoplasia (CIN) from normal tissue by means of two different fluorescence modalities. Most of the patients were given a low dose (5 mg/kg bw) of (delta) -amino levulinic acid (ALA). The ALA was orally administered 2 - 4 hours prior to the investigation. During this time, the ALA is transformed to the strongly fluorescent protoporphyrin IX (PpIX) via the haem cycle. Excitation light with a wavelength of 405 nm was used to excite the PpIX fluorescence. Excess amounts of PpIX were accumulated preferentially in diseased tissue. However, the variability in the PpIX accumulation from patient to patient was large. By using excitation light at 337 nm, the endogenous fluorophores are more efficiently excited. Therefore, this excitation modality was exploited for studying spectral characteristics of the autofluorescence in different tissue types. The spectra obtained were evaluated by forming fluorescence intensity ratios. The tissue types were grouped according to the histopathological examination. A correlation with the fluorescence ratios was performed. Some problems with the classification remain, mostly due to the difficulties in obtaining histopathologic evaluation of the biopsies at the exact location of the LIF measurements.

Analysis of spatial variability in hyperspectral imagery of the uterine cervix in vivo

The use of fluorescence and reflectance spectroscopy in the analysis of cervical histopathology is a growing field of research. The majority of this research is performed with point-like probes. Typically, clinicians select probe sites visually, collecting a handful of spectral samples. An exception to this methodology is the Hyperspectral Diagnostic Imaging (HSDI) instrument developed by Science and Technology International. This non-invasive device collects contiguous hyperspectral images across the entire cervical portio. The high spatial and spectral resolution of the HSDI instruments make them uniquely well suited for addressing the issues of coupled spatial and spectral variability of tissues in vivo. Analysis of HSDI data indicates that tissue spectra vary from point to point, even within histopathologically homogeneous regions. This spectral variability exhibits both random and patterned components, implying that point monitoring may be susceptible to significant sources of noise and clutter inherent in the tissue. We have analyzed HSDI images from clinical CIN (cervical intraepithelial neoplasia) patients to quantify the spatial variability of fluorescence and reflectance spectra. This analysis shows the spatial structure of images to be fractal in nature, in both intensity and spectrum. These fractal tissue textures will limit the performance of any point-monitoring technology.

In-vivo fluorescence and reflectance imaging of human cervical tissue

A hyperspectral imaging spectrograph has been used to measure the fluorescence and reflectance of cervical tissue in vivo. The instrument was employed in a clinical trial in Vilnius, Lithuania, where 111 patients were examined. The patients were initially screened by Pap smear, examined by colposcopy and a tissue sampling procedure was performed. Detailed histopathological assessments were performed on the biopsies, and these assessments were correlated with spectra and images. The results of the spectroscopic investigations show that different tissue types within one biopsy region exhibit different spectral signatures. A spectral analysis of the entire image localizes dysplastic regions in both fluorescence and reflectance, suggesting that the hyperspectral imaging technique is useful in the management of cervical malignancies.

Spinal hernia tissue autofluorescence spectrum

The laser intervertebral disc decompression may provide appropriate relief in properly selected patients with contained disc herniations. The present investigation aims to characterise intervertebral disc material by autofluorescence induced by laser light. Degeneration of the intervertebral disc is associated with progressive biochemical changes in disc material. Percutaneous laser disc decompression has become rather popular for the treatment of lumbar disc herniation, but there are problems in the selection of patients. For this purpose, recognition of the disc composition is necessary. We propose a new type of spectroscopic investigation. It is advantageous to the characterization of intervertebral disc material. Intervertebral disc specimens were removed during open surgery from different disc locations. Preoperative patients’ MRI was evaluated using the Pfirrmann disc degeneration and Komori scale for migrating of herniated nucleus pulposus. Adjacent slices of stained disc sections were evaluated by histology/histochemistry and autofluorescence spectra. Comparison of the MRI, spectral, histological and histochemical data was performed. The MRI Komori scale correlated with the histology Boos degeneration index. In the histochemistry, collagens other than collagens I and II of the disc were distinguished with best positive correlation coefficient (0.829) and best negative one (−0.904) of proteoglycans of sequester to Boos index. A correlation of the IV Gaussian component of the hernia spectra with the Boos index was established. The Gaussian component correlation with different collagen types and proteoglycan was determined for the disc and sequester. “Autofluorescence-based diagnosis” refers to the evaluation of disc degeneration by histological and histochemical evaluation; it can provide additional data on the degeneration of an intervertebral disc.

Cervical Smear Photodiagnosis by Fluorescence

OBJECTIVE Fluorescence is widely investigated and can characterize general changes occurring in the state of the cell and the tissue during physiological and/or pathological processes, but the method has not been adopted for clinical use. We present a photodiagnostic device and a relevant evaluation method fitted to classical screening procedures. This method is based on the discovery of smear content intrinsic fluorescent markers. BACKGROUND DATA Meaningful spectral components of cervical smear samples differ from those measured in the entire live cervix. This article deals with the identification of changes in smear spectra in cervicitis and CIN2+ (cervical intraepithelial neoplasia of the second degree or higher) at 355 nm excitation. METHODS Methods used in the study: microlaser-induced fluorescence spectroscopy of liquid cytology samples and histological evaluation of the biopsies of the same cervix (and/or only cytological evaluation) was performed for 78 cases. The fluorescence spectra of cervical cytology supernatant sediment were approximated with several Gaussian components. The ratios of the area under each Gaussian component to the whole area under the experimental curve were calculated and compared among histological groups by using the Mann-Whitney test and receiver operating characteristic (ROC) analysis. RESULTS The results of this study are a concise summary of the essential features verified by the data: the spectral regions 402-416 nm and 424-438 nm are important for the discrimination of both normal and CIN2+ groups (in terms of sensitivity, specificity, and positive predictive value). The spectral regions 480-515 nm and 595-625 nm are important for the identification of cervicitis. CONCLUSIONS Cervical smear autofluorescence diagnostics could be useful for cancer screening at the point of care, in a simple cytology laboratory, and for the monitoring of treatment. We suggest possible fluorophores in the smear content.

Optimal Spectral Regions For Laser Excited Fluorescence Diagnostics For Point Of Care Application

The tissue fluorescence gives the response of light emitting molecule signature, and characterizes the cell composition and peculiarities of metabolism. Both are useful for the biomedical diagnostics, as reported in previous our and others works. The present work demonstrates the results of application of laser excited autofluorescence for diagnostics of pathology in genital tissues, and the feasibility for the bedside at “point of care—off lab” application. A portable device using the USB spectrophotometer, micro laser (355 nm Nd:YAG, 0,5 ns pulse, repetition rate 10 kHz, output power 15 mW), three channel optical fiber and computer with diagnostic program was designed and ready for clinical trial to be used for cytology and biopsy specimen on site diagnostics, and for the endoscopy/puncture procedures. The biopsy and cytology samples, as well as intervertebral disc specimen were evaluated by pathology experts and the fluorescence spectra were investigated in the fresh and preserved specimens. The spectr...

FLUORESCENCE SPECTRUM AND DECAY MEASUREMENT FOR HSIL VS NORMAL CYTOLOGY DIFFERENTIATION IN LIQUID PAP SMEAR SUPERNATANT

Cervical smear material contains endo and exocervical cells, mucus and inflammative, immune cells in cases of pathology. Just not destroyed keratinocytes lay on the glass for microscopy. Liquid cytology supernatant apart other diagnostics could be used for photodiagnostic. The spectroscopic parameters suitable for Normal and HSIL cytology groups supernatant differentiation are demonstrated.The dried liquid PAP supernatant fractions—sediment and liquid were investigated. Excitation and emission matrices (EEM), supernatant fluorescence decay measured under 280 nm diode short pulse excitation and fluorescence spectroscopy by excitation with 355 nm laser light were analyzed.The differences between Normal and HSIL groups were statistically proven in the certain spectral regions. Fluorescence decay peculiarities show spectral regions consisting of few fluorophores. Obtained results on fluorescence differences in Normal and HSIL groups’ supernatant shows the potency of photodiagnosis application in cervical scre...

Analysis of fluorescence excitation emission matrices of endometrial tissue

We have analysed an endometrial tissue fluorescence excitation spectra with a purpose to determine the characteristic wavelength of excitation for multiwavelength diagnostics of tissue. Fluorescence excitation-emission matrices from 200 to 500 nm excitation and 200 to 800 nm emission were measured as a supplementary data for previous measurements to unambiguously characterize biochemicals quantitatively in endometrial tissue.

Spectroscopic Parameters of Lumbar Intervertebral Disc Material

There are numerous methods of investigating intervertebral disc. Visualization methods are widely used in clinical practice. Histological, imunohistochemical and biochemical methods are more used in scientific research. We propose that a new spectroscopic investigation would be useful in determining intervertebral disc material, especially when no histological specimens are available.Purpose: to determine spectroscopic parameters of intervertebral disc material; to determine emission spectra common for all intervertebral discs; to create a background for further spectroscopic investigation where no histological specimen will be available.Material and Methods: 20 patients, 68 frozen sections of 20 μm thickness from operatively removed intervertebral disc hernia were excited by Nd:YAG microlaser STA‐01‐TH third harmonic 355 nm light throw 0, 1 mm fiber. Spectrophotometer OceanOptics USB2000 was used for spectra collection. Mathematical analysis of spectra was performed by ORIGIN multiple Gaussian peaks anal...

Hypericin-based photodynamic therapy: antitumor activity, accumulation potential, and induced cell death pathway

In this study the main interest was focused on the to investigation the photodynamic efficacy of hypericin, three other photosensitizers and 5 aminolevulinic acid-induced protopofirin IX in their ability to block the growth of rather aggressive tumor - Ehrlich ascite carcinoma in mice as well as Reh cells in humans (B-leukemia). Hypericin was found to exhibit the highest phototoxicity and antitumor activity in treating Ehrlich ascite carcinoma. The different photosensitizers were ranked as follows: Hypericin > hematoporphyrin dimethyl ether > Photofrin II > meso-tetra (para-sulfophenyl)porphin > 5-aminolevulinic acid. The most important is that just after Hyp-based photodynamic therapy 75% of mice survived a 4 month-period, and no recurrence of tumor within this period was detected in 25% of the treated mice. The clear cut correlation observed between intracellular dye concentration in the tumor cells and efficiency of photodynamic therapy, supports the idea that the intracellular accumulation of the photosensitizer is one of the most important factors in determining the benefit of photodynamic therapy. Hence, the accumulation of the photosensitizer in the tumor cells should be considered as one of the prognostic factors for the determination of the therapeutic outcome. Eventually, one of the most significant result is that hypericin is effective photosensitizer for human B-leukemia cells and induces apoptosis after photosensitization.